Adenoviral vectors transiently exhibit high efficiency of gene transfer and expression as a vector for introducing genes into mammalian cells. They, furthermore, allow gene transfer to non-dividing cells and enable expression through administration to animal subjects, therefore, they have been developed as vectors for gene therapy (Breyer et al., Current Gene Therapy, Vol. 1, pp. 149–162). Although it is known that the adenoviral vectors have wide host range, the efficiency of gene transfer with regard to blood cells is low (Marini III et al., Cancer Gene Therapy, Vol. 7, pp. 816–825).
Infection of adenovirus into cells is thought to be a process consisting of two-stages: adsorption and internalization. It is thought that human adenovirus type 5 used as a vector, is adsorbed to a cell due to binding between a fiber of the virus and a cell surface protein, CAR (coxsackievirus adenovirus receptor) (Bergelson et al., Science, Vol. 275, 1320–1323), and then it is internalized into the cell due to binding between a penton base of the virus and cell surface proteins, integrins (Wickham et al., Cell, Vol. 73, pp. 309–319). The efficiency of adenoviral vector-mediated gene transfer is dependent on the intensity of the CAR expression of the target cells. However, CAR expression of blood cells is generally low, and in particular, it has been shown that undifferentiated blood cells, CD34 positive and CD38 negative cells hardly express CAR at all (Rebel et al., Stem Cells, Vol. 18, pp. 176–182). Further, a separate report (Leon et al., Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 13159–13164) indicates that with lymphocytes that are forced to express CAR, the efficiency of adenoviral vector-mediated gene transfer improves. Therefore, the problem of the low efficiency of gene transfer to blood cells is caused by difficulty in adsorption of the virus to the cells. Increasing adsorption is an important technical target for improving efficiency of adenoviral vector-mediated gene transfer to blood cells, and various approaches directed to this object have been reported.
Smith et al. reported that they prepared a specific host-retargeting adenoviral vector by binding a ligand molecule being any one of SCF (stem cell factor), anti-c-Kit antibody, anti-CD34 antibody, anti-CD44 antibody or IL-2 to an adenovirus particle using avidin-biotin binding method, and that the modified vectors were improved in the efficiency of gene transfer to blood cells that expressed these specific receptors (Proc. Natl. Acad. Sci. USA, Vol. 96, pp. 8855–8860). However, according to the report of Smith et al., with M07e cells infected by this method with luciferase or GFP (green fluorescent protein) as a reporter gene, 95% positive cells were detected by PCR (polymerase chain reaction) on the virus genome DNA, but in the case of detection of the level of expression of GFP protein by flow cytometer, positive cells were slightly less that 20%, and it has been pointed out that there is a problem with internalization of the virus or in the expression stage of the virus genome.
Shayakhmetov et al. (J. Virol., Vol. 74, pp. 2567–2583) and Yotnda et al. (Gene Therapy, Vol. 8, pp. 930–937) reported that they substituted only fibers of an adenovirus type 5 vector with those of adenovirus type 35, developing an Ad5/F35 vector having different tropism to that of adenovirus type 5, and that the Ad5/F35 vector was improved in the efficiency of gene transfer to blood cells. This method requires a virus genome to be newly prepared, and it should be noted that a receptor molecule of adenovirus type 35 has not been identified. Thus, there is the problem that safety or pathogenicity in respect of human adenovirus type 35 has not been clarified.
On the other hand, there have been reports that gene transfer to blood cells is possible even where an ordinary adenovirus vector is used without carrying out any special modifications. (Watanabe et al., Blood, Vol. 87, pp. 5032–5039; Neering et al., Blood, Vol. 88, pp. 1147–1155; Takahashi et al, Blood, Vol. 91, pp. 4509–4515; Fan et al., Hum. Gene Ther, Vol. 11, pp. 1313–1327). In these reports, virus infection was performed at a high MOI (multiplicity of infection) of 100–500 and over a long period of 24 to 48 hours, and cell adsorption of the virus is thought to be non-specific (Marini IIIrd et al., Cancer Gene Therapy, Vol. 7, pp. 816–825). General adenovirus vector infection conditions are around 2 hours at about MOI 50, however, it is well known that adenovirus infection at a high concentration causes cytotoxicity, and it has been shown that toxicity is high particularly for undifferentiated blood cells (MacKenzie et al., Blood, Vol. 96, pp. 100–108).
Further, to promote introduction of genes into the target cells of nucleic acid delivery vehicles such as virus vectors, etc., there has been disclosed a complex construct of (1) a molecule which binds to the nucleic acid delivery vehicle and (2) a molecule which binds to the surface molecules of target cells linked by (3) a linker (O'Riordan et al., WO99/40214 and U.S. Pat. No. 6,287,857). In a concrete example of this, a complex construct can be used in which antibodies or peptides having binding activity corresponding to each of the nucleic acid delivery vehicles are chemically bound to surface molecules of target cells. Because the method of producing this complex construct involves chemically binding two different peptides using a coupling reagent, there are thought to be problems such as low product yield and non-uniform structure of the products.
There has also been reported a method that uses a polypeptide consisting of two constituent factors, an extracellular portion of CAR protein and a soluble ligand having affinity for target cells, as an agent to assist adsorption. Dmitriev et al. prepared a fusion polypeptide of CAR and epidermal growth factor, EGF, and reported that this polypeptide promotes infection of adenovirus to tumor cells expressing EGF receptor (J. Virol, Vol. 74, pp. 6875–6884). Further, Ebbinghaus et al. prepared a fusion polypeptide of CAR and human immunoglobulin Fc region, and reported promotion of infection of adenovirus to cells expressing Fcγ receptor (J. Virol, Vol. 75, pp. 480–489). However, neither EGF receptor nor Fcγ receptor is expressed in undifferentiated blood cells, and these fusion polypeptides cannot be applied to undifferentiated blood cells.
Therefore, there is a need to provide a specific adsorption agent for adenovirus 5 to undifferentiated blood cells that allows virus infection at an MOI that does not impart toxicity to undifferentiated blood cells, without requiring construction of a new virus genome, and without requiring special modifications such as biotination of the virus particle.